Selective glycosidase inhibitors and uses thereof

ABSTRACT

The invention provides compounds for selectively inhibiting glycosidases, prodrugs of the compounds, and pharmaceutical compositions including the compounds or prodrugs of the compounds. The invention also provides methods of treating diseases and disorders related to deficiency or overexpression of O-GlcNAcase, accumulation or deficiency of O-GlcNAc.

FIELD OF THE INVENTION

This application relates to compounds which selectively inhibitglycosidases and uses thereof.

BACKGROUND OF THE INVENTION

A wide range of cellular proteins, both nuclear and cytoplasmic, arepost-translationally modified by the addition of the monosaccharide2-acetamido-2-deoxy-β-D-glucopyranoside (β-N-acetylglucosamine) which isattached via an β-glycosidic linkage.¹ This modification is generallyreferred to as O-linked N-acetylglucosamine or O-GlcNAc. The enzymeresponsible for post-translationally linking β-N-acetylglucosamine(GlcNAc) to specific serine and threonine residues of numerousnucleocytoplasmic proteins is O-GlcNAc transferase (OGT).²⁻⁵ A secondenzyme, known as glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidase(O-GlcNAcase)^(6,7) removes this post-translational modification toliberate proteins making the O-GlcNAc-modification a dynamic cycleoccurring several times during the lifetime of a protein.⁸

O-GlcNAc-modified proteins regulate a wide range of vital cellularfunctions including, for example, transcription,⁹⁻¹² proteasomaldegradation,¹³ and cellular signaling.¹⁴ O-GlcNAc is also found on manystructural proteins.¹⁵⁻¹⁷ For example, it has been found on a number ofcytoskeletal proteins, including neurofilament proteins,^(18,19)synapsins,^(6,20) synapsin-specific clathrin assembly protein AP-3,⁷ andankyrinG.¹⁴ O-GlcNAc modification has been found to be abundant in thebrain.^(21,22) It has also been found on proteins clearly implicated inthe etiology of several diseases including Alzheimer's disease (AD) andcancer.

For example, it is well established that AD and a number of relatedtauopathies including Downs' syndrome, Pick's disease, Niemann-Pick TypeC disease, and amyotrophic lateral sclerosis (ALS) are characterized, inpart, by the development of neurofibrillary tangles (NFTs). These NFTsare aggregates of paired helical filaments (PHFs) and are composed of anabnormal form of the cytoskeletal protein “tau”. Normally tau stabilizesa key cellular network of microtubules that is essential fordistributing proteins and nutrients within neurons. In AD patients,however, tau becomes hyperphosphorylated, disrupting its normalfunctions, forming PHFs and ultimately aggregating to form NFTs. Sixisoforms of tau are found in the human brain. In AD patients, all sixisoforms of tau are found in NFTs, and all are markedlyhyperphosphorylated.^(23,24) Tau in healthy brain tissue bears only 2 or3 phosphate groups, whereas those found in the brains of AD patientsbear, on average, 8 phosphate groups.^(25,26) A clear parallel betweenNFT levels in the brains of AD patients and the severity of dementiastrongly supports a key role for tau dysfunction in AD.^(27,28) Theprecise causes of this hyperphosphorylation of tau remain elusive.Accordingly, considerable effort has been dedicated toward: a)elucidating the molecular physiological basis of tauhyperphosphorylation;²⁹ and b) identifying strategies that could limittau hyperphosphorylation in the hope that these might halt, or evenreverse, the progression of Alzheimer's disease³⁰⁻³³ Thus far, severallines of evidence suggest that up-regulation of a number of kinases maybe involved in hyperphosphorylation of tau,^(21,34,35) although veryrecently, an alternative basis for this hyperphosphorylation has beenadvanced.²¹

In particular, it has emerged that phosphate levels of tau are regulatedby the levels of O-GlcNAc on tau. The presence of O-GlcNAc on tau hasstimulated studies that correlate O-GlcNAc levels with tauphosphorylation levels. The interest in this field stems from theobservation that O-GlcNAc modification has been found to occur on manyproteins at amino acid residues that are also known to bephosphorylated.³⁶⁻³⁸ Consistent with this observation, it has been foundthat increases in phosphorylation levels result in decreased O-GlcNAclevels and conversely, increased O-GlcNAc levels correlate withdecreased phosphorylation levels.³⁹ This reciprocal relationship betweenO-GlcNAc and phosphorylation has been termed the “Yin-Yang hypothesis”⁴⁰and has gained strong biochemical support by the discovery that theenzyme OGT⁴ forms a functional complex with phosphatases that act toremove phosphate groups from proteins.⁴¹ Like phosphorylation, O-GlcNAcis a dynamic modification that can be removed and reinstalled severaltimes during the lifespan of a protein. Suggestively, the gene encodingO-GlcNAcase has been mapped to a chromosomal locus that is linked toAD.^(7,42) Hyperphosphorylated tau in human AD brains has markedly lowerlevels of O-GlcNAc than are found in healthy human brains.²¹ It has beenshown that O-GlcNAc levels of soluble tau protein from human brainsaffected with AD are markedly lower than those from healthy brain.²¹Furthermore, PHF from diseased brain was suggested to lack completelyany O-GlcNAc modification whatsoever.²¹ The molecular basis of thishypoglycosylation of tau is not known, although it may stem fromincreased activity of kinases and/or dysfunction of one of the enzymesinvolved in processing O-GlcNAc. Supporting this latter view, in bothPC-12 neuronal cells and in brain tissue sections from mice, anonselective N-acetylglucosamindase inhibitor was used to increase tauO-GlcNAc levels, whereupon it was observed that phosphorylation levelsdecreased.²¹ The implication of these collective results is that bymaintaining healthy O-GlcNAc levels in AD patients, such as byinhibiting the action of O-GlcNAcase, one should be able to blockhyperphosphorylation of tau and all of the associated effects of tauhyperphosphorylation, including the formation of NFTs and downstreameffects. However, because the proper functioning of theβ-hexosaminidases is critical, any potential therapeutic interventionfor the treatment of AD that blocks the action of O-GlcNAcase would haveto avoid the concomitant inhibition of both hexosaminidases A and B.

Neurons do not store glucose and therefore the brain relies on glucosesupplied by blood to maintain its essential metabolic functions.Notably, it has been shown that within brain, glucose uptake andmetabolism decreases with aging.⁴³ Within the brains of AD patientsmarked decreases in glucose utilization occur and are thought to be apotential cause of neurodegeneration.⁴⁴ The basis for this decreasedglucose supply in AD brain⁴⁵⁻⁴⁷ is thought to stem from any of decreasedglucose transport,^(48,49) impaired insulin signaling,^(50,51) anddecreased blood flow.⁵²

In light of this impaired glucose metabolism, it is worth noting that ofall glucose entering into cells, 2-5% is shunted into the hexosaminebiosynthetic pathway, thereby regulating cellular concentrations of theend product of this pathway, uridine diphosphate-N-acetylglucosamine(UDP-GlcNAc).⁵³ UDP-GlcNAc is a substrate of the nucleocytoplasmicenzyme O-GlcNAc transferase (OGT),²⁻⁵ which acts to post-translationallyadd GlcNAc to specific serine and threonine residues of numerousnucleocytoplasmic proteins. OGT recognizes many of itssubstrates^(54,55) and binding partners^(41,56) through itstetratricopeptide repeat (TPR) domains.^(57,58) As described above,O-GlcNAcase^(6,7) removes this post-translational modification toliberate proteins making the O-GlcNAc-modification a dynamic cycleoccurring several times during the lifetime of a protein.⁸ O-GlcNAc hasbeen found in several proteins on known phosphorylationsites,^(10,37,38,59) including tau and neurofilaments.⁶⁰ Additionally,OGT shows unusual kinetic behaviour making it exquisitely sensitive tointracellular UDP-GlcNAc substrate concentrations and therefore glucosesupply.⁴¹

Consistent with the known properties of the hexosamine biosyntheticpathway, the enzymatic properties of OGT, and the reciprocalrelationship between O-GlcNAc and phosphorylation, it has been shownthat decreased glucose availability in brain leads to tauhyperphosphorylation.⁴⁴ Therefore the gradual impairment of glucosetransport and metabolism, whatever its causes, leads to decreasedO-GlcNAc and hyperphosphorylation of tau (and other proteins).Accordingly, the inhibition of O-GlcNAcase should compensate for the agerelated impairment of glucose metabolism within the brains of healthindividuals as well as patients suffering from AD or relatedneurodegenerative diseases.

These results suggest that a malfunction in the mechanisms regulatingtau O-GlcNAc levels may be vitally important in the formation of NFTsand associated neurodegeneration. Good support for blocking tauhyperphosphorylation as a therapeutically useful intervention⁶¹ comesfrom recent studies showing that when transgenic mice harbouring humantau are treated with kinase inhibitors, they do not develop typicalmotor defects³³ and, in another case,³² show decreased levels ofinsoluble tau. These studies provide a clear link between lowering tauphosphorylation levels and alleviating AD-like behavioural symptoms in amurine model of this disease. Indeed, pharmacological modulation of tauhyperphosphorylation is widely recognized as a valid therapeuticstrategy for treating AD and other neurodegenerative disorders.⁶²

Small-molecule O-GlcNAcase inhibitors, to limit tauhyperphosphorylation, have been considered for treatment of AD andrelated tauopathies⁶³. Specifically, the O-GlcNAcase inhibitor thiamet-Ghas been implicated in the reduction of tau phosphorylation in culturedPC-12 cells at pathologically relevant sites.⁶³ Moreover, oraladministration of thiamet-G to healthy Sprague-Dawley rats has beenimplicated in reduced phosphorylation of tau at Thr231, Ser396 andSer422 in both rat cortex and hippocampus.⁶³

There is also a large body of evidence indicating that increased levelsof O-GlcNAc protein modification provides protection against pathogeniceffects of stress in cardiac tissue, including stress caused byischemia, hemorrhage, hypervolemic shock, and calcium paradox. Forexample, activation of the hexosamine biosynthetic pathway (HBP) byadministration of glucosamine has been demonstrated to exert aprotective effect in animals models of ischemia/reperfusion,⁶⁴⁻⁷⁰ traumahemorrhage,⁷¹⁻⁷³ hypervolemic shock,⁷⁴ and calcium paradox.^(64,75)Moreover, strong evidence indicates that these cardioprotective effectsare mediated by elevated levels of protein O-GlcNAcmodification.^(64,65,67,70,72,75-78) There is also evidence that theO-GlcNAc modification plays a role in a variety of neurodegenerativediseases, including Parkinson's disease and Huntington's disease.⁷⁹

Humans have three genes encoding enzymes that cleave terminalβ-N-acetyl-glucosamine residues from glycoconjugates. The first of theseencodes O-GlcNAcase. O-GlcNAcase is a member of family 84 of glycosidehydrolases that includes enzymes from organisms as diverse asprokaryotic pathogens to humans (for the family classification ofglycoside hydrolases see Coutinho, P. M. & Henrissat, B. (1999)Carbohydrate-Active Enzymes server at URL:http://afmb.cnrs-mrs.fr/CAZY/.^(27,28) O-GlcNAcase acts to hydrolyseO-GlcNAc off of serine and threonine residues of post-translationallymodified proteins.^(1,6,7,80,81) Consistent with the presence ofO-GlcNAc on many intracellular proteins, the enzyme O-GlcNAcase appearsto have a role in the etiology of several diseases including type IIdiabetes,^(14,82) AD,^(16,21,83) and cancer.^(22,84) AlthoughO-GlcNAcase was likely isolated earlier on,^(18,19) about 20 yearselapsed before its biochemical role in acting to cleave O-GlcNAc fromserine and threonine residues of proteins was understood.⁶ More recentlyO-GlcNAcase has been cloned,⁷ partially characterized,²⁰ and suggestedto have additional activity as a histone acetyltransferase.²⁰ However,little was known about the catalytic mechanism of this enzyme.

The other two genes, HEXA and HEXB, encode enzymes catalyzing thehydrolytic cleavage of terminal β-N-acetylglucosamine residues fromglycoconjugates. The gene products of HEXA and HEXB predominantly yieldtwo dimeric isozymes, hexosaminidase A and hexosaminidase B,respectively. Hexosaminidase A (αβ), a heterodimeric isozyme, iscomposed of an α- and a β-subunit. Hexosaminidase B (ββ), a homodimericisozyme, is composed of two β-subunits. The two subunits, α- and β-,bear a high level of sequence identity. Both of these enzymes areclassified as members of family 20 of glycoside hydrolases and arenormally localized within lysosomes. The proper functioning of theselysosomal β-hexosaminidases is critical for human development, a factthat is underscored by the tragic genetic illnesses, Tay-Sach's andSandhoff diseases which stem from a dysfunction in, respectively,hexosaminidase A and hexosaminidase B.⁸⁵ These enzymatic deficienciescause an accumulation of glycolipids and glycoconjugates in thelysosomes resulting in neurological impairment and deformation. Thedeleterious effects of accumulation of gangliosides at the organismallevel are still being uncovered.⁸⁶

As a result of the biological importance of theseβ-N-acetyl-glucosaminidases, small molecule inhibitors ofglycosidases⁸⁷⁻⁹⁰ have received a great deal of attention,⁹¹ both astools for elucidating the role of these enzymes in biological processesand in developing potential therapeutic applications. The control ofglycosidase function using small molecules offers several advantagesover genetic knockout studies including the ability to rapidly varydoses or to entirely withdraw treatment.

However, a major challenge in developing inhibitors for blocking thefunction of mammalian glycosidases, including O-GlcNAcase, is the largenumber of functionally related enzymes present in tissues of highereukaryotes. Accordingly, the use of non-selective inhibitors in studyingthe cellular and organismal physiological role of one particular enzymeis complicated because complex phenotypes arise from the concomitantinhibition of such functionally related enzymes. In the case ofβ-N-acetylglucosaminidases, many compounds that act to block O-GlcNAcasefunction are non-specific and act potently to inhibit thelysosomal-hexosaminidases.

A few of the better characterized inhibitors ofβ-N-acetyl-glucosaminidases which have been used in studies of O-GlcNAcpost-translational modification within both cells and tissues arestreptozotocin (STZ), 2′-methyl-α-D-glucopyrano-[2,1-d]-Δ2′-thiazoline(NAG-thiazoline) and O-(2-acetamido-2-deoxy-D-glucopyranosylidene)aminoN-phenylcarbamate (PUGNAc).^(14,92-95)

STZ has long been used as a diabetogenic compound because it has aparticularly detrimental effect on β-islet cells.⁹⁶ STZ exerts itscytotoxic effects through both the alkylation of cellular DNA^(96,97) aswell as the generation of radical species including nitric oxide.⁹⁸ Theresulting DNA strand breakage promotes the activation ofpoly(ADP-ribose) polymerase (PARP)⁹⁹ with the net effect of depletingcellular NAD+ levels and, ultimately, leading to cell death.^(100,101)Other investigators have proposed instead that STZ toxicity is aconsequence of the irreversible inhibition of O-GlcNAcase, which ishighly expressed within β-islet cells.^(92,102) This hypothesis has,however, been brought into question by two independent researchgroups.^(103, 104) Because cellular O-GlcNAc levels on proteins increasein response to many forms of cellular stress¹⁰⁵ it seems possible thatSTZ results in increased O-GlcNAc-modification levels on proteins byinducing cellular stress rather than through any specific and directaction on O-GlcNAcase. Indeed, Hanover and coworkers have shown that STZfunctions as a poor and somewhat selective inhibitor of O-GlcNAcase¹⁰⁶and although it has been proposed by others that STZ acts toirreversibly inhibit O-GlcNAcase,¹⁰⁷ there has been no cleardemonstration of this mode of action. More recently, it has been shownthat STZ does not irreversibly inhibit O-GlcNAcase.¹⁰⁸

NAG-thiazoline has been found to be a potent inhibitor of family 20hexosaminidases,^(90,109) and more recently, the family 84O-GlcNAcases.¹⁰⁸ Despite its potency, a downside to using NAG-thiazolinein a complex biological context is that it lacks selectivity andtherefore perturbs multiple cellular processes.

PUGNAc is another compound that suffers from the same problem of lack ofselectivity, yet has enjoyed use as an inhibitor of both humanO-GlcNAcase^(6,110) and the family 20 human β-hexosaminidases.¹¹¹ Thismolecule, developed by Vasella and coworkers, was found to be a potentcompetitive inhibitor of the β-N-acetyl-glucosaminidases from Canavaliaensiformis, Mucor rouxii, and the β-hexosaminidase from bovine kidney.⁸⁸It has been demonstrated that administration of PUGNAc in a rat model oftrauma hemorrhage decreases circulating levels of the pro-inflammatorycytokines TNF-α and IL-6.¹¹² It has also been shown that administrationof PUGNAc in a cell-based model of lymphocyte activation decreasesproduction of the cytokine IL-2.¹¹³ Subsequent studies have indicatedthat PUGNAc can be used in an animal model to reduce myocardial infarctsize after left coronary artery occlusions.¹¹⁴ Of particularsignificance is the fact that elevation of O-GlcNAc levels byadministration of PUGNAc, an inhibitor of O-GlcNAcase, in a rat model oftrauma hemorrhage improves cardiac function.^(112,115) In addition,elevation of O-GlcNAc levels by treatment with PUGNAc in a cellularmodel of ischemia/reperfusion injury using neonatal rat ventricularmyocytes improved cell viability and reduced necrosis and apoptosiscompared to untreated cells.¹¹⁶

More recently, it has been suggested that the selective O-GlcNAcaseinhibitor NButGT exhibits protective activity in cell-based models ofischemia/reperfusion and cellular stresses, including oxidativestress.¹¹⁷ This study suggests the use of O-GlcNAcase inhibitors toelevate protein O-GlcNAc levels and thereby prevent the pathogeniceffects of stress in cardiac tissue.

International patent applications PCT/CA2006/000300, filed 1 Mar. 2006,published under No. WO 2006/092049 on 8 Sep. 2006; PCT/CA2007/001554,filed 31 Aug. 2007, published under No. WO 2008/025170 on 6 Mar. 2008;PCT/CA2009/001087, filed 31 Jul. 2009, published under No. WO2010/012106 on 4 Feb. 2010; PCT/CA2009/001088, filed 31 Jul. 2009,published under WO 2010/012107 on 4 Feb. 2010; and PCT/CA2009/001302,filed 16 Sep. 2009, published under WO 2010/037207 on 8 Apr. 2010,describe selective inhibitors of O-GlcNAcase.

SUMMARY OF THE INVENTION

The invention provides, in part, compounds for selectively inhibitingglycosidases, prodrugs of the compounds, uses of the compounds and theprodrugs, pharmaceutical compositions including the compounds orprodrugs of the compounds, and methods of treating diseases anddisorders related to deficiency or overexpression of O-GlcNAcase, and/oraccumulation or deficiency of O-GlcNAc.

In one aspect, the invention provides a compound of Formula (I) or apharmaceutically acceptable salt thereof:

where each R¹ may be independently a non-interfering substituentselected from H or acyl; each R² may be independently alkyl, acyl, oralkoxy; R³ may be OR⁴ or NR⁴ ₂; wherein each R⁴ may be optionallyindependently a non-interfering substituent selected from H, alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, acyl, or carbamoyl; andwherein two R² groups are connected together with the nitrogen atom towhich they are attached to form a ring.

In alternative embodiments, the non-interfering substituent may includeone or more heteroatoms selected from P, O, S, N, F, Cl, Br, I, or B.The non-interfering substituent may be optionally substituted.

In alternative embodiments, the invention provides a compound of Formula(I) or a pharmaceutically acceptable salt thereof:

where each R¹ may be independently H or C₁₋₆ acyl; both R² groups may bejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ may be OR⁴ or NR⁴ ₂; wherein each R⁴ may beindependently selected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, orcarbamoyl.

In alternative embodiments, the compound may be a prodrug; the compoundmay selectively inhibit an O-glycoprotein2-acetamido-2-deoxy-(3-D-glucopyranosidase (O-GlcNAcase); the compoundmay selectively bind an O-GlcNAcase (e.g., a mammalian O-GlcNAcase); thecompound may selectively inhibit the cleavage of a2-acetamido-2-deoxy-β-D-glucopyranoside (O-GlcNAc); the compound may notsubstantially inhibit a mammalian β-hexosaminidase.

In alternative aspects, the invention provides a pharmaceuticalcomposition including a compound according to the invention, incombination with a pharmaceutically acceptable carrier.

In alternative aspects, the invention provides methods of selectivelyinhibiting an O-GlcNAcase, or of inhibiting an O-GlcNAcase in a subjectin need thereof, or of increasing the level of O-GlcNAc, or of treatinga neurodegenerative disease, a tauopathy, cancer or stress, in a subjectin need thereof, by administering to the subject an effective amount ofa compound of Formula (I) or a pharmaceutically acceptable salt thereof:

where each R¹ may be independently H or C₁₋₆ acyl; both R² groups may bejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ may be OR⁴ or NR⁴ ₂; wherein each R⁴ may beindependently selected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, orcarbamoyl. The condition may be Alzheimer's disease, Amyotrophic lateralsclerosis (ALS), Amyotrophic lateral sclerosis with cognitive impairment(ALSci), Argyrophilic grain dementia, Bluit disease, Corticobasaldegeneration (CBD), Dementia pugilistica, Diffuse neurofibrillarytangles with calcification, Down's syndrome, Familial British dementia,Familial Danish dementia, Frontotemporal dementia with parkinsonismlinked to chromosome 17 (FTDP-17), Gerstmann-Straussler-Scheinkerdisease, Guadeloupean parkinsonism, Hallevorden-Spatz disease(neurodegeneration with brain iron accumulation type 1), Multiple systematrophy, Myotonic dystrophy, Niemann-Pick disease (type C),Pallido-ponto-nigral degeneration, Parkinsonism-dementia complex ofGuam, Pick's disease (PiD), Post-encephalitic parkinsonism (PEP), Priondiseases (including Creutzfeldt-Jakob Disease (CJD), VariantCreutzfeldt-Jakob Disease (vCJD), Fatal Familial Insomnia, and Kuru),Progressive supercortical gliosis, Progressive supranuclear palsy (PSP),Richardson's syndrome, Subacute sclerosing panencephalitis, Tangle-onlydementia, Huntington's disease, Parkinson's disease, Schizophrenia, MildCognitive Impairment (MCI), Neuropathy (including peripheral neuropathy,autonomic neuropathy, neuritis, and diabetic neuropathy), or Glaucoma.The stress may be a cardiac disorder, e.g., ischemia; hemorrhage;hypovolemic shock; myocardial infarction; an interventional cardiologyprocedure; cardiac bypass surgery; fibrinolytic therapy; angioplasty; orstent placement.

In alternative aspects, the invention provides a method of treating anO-GlcNAcase-mediated condition that excludes a neurodegenerativedisease, a tauopathy, cancer or stress, in a subject in need thereof, byadministering to the subject an effective amount of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof:

where each R¹ may be independently H or C₁₋₆ acyl; both R² groups may bejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ may be OR⁴ or NR⁴ ₂; wherein each R⁴ may beindependently selected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, orcarbamoyl. In some embodiments, the condition may be inflammatory orallergic diseases such as asthma, allergic rhinitis, hypersensitivitylung diseases, hypersensitivity pneumonitis, eosinophilic pneumonias,delayed-type hypersensitivity, atherosclerosis, interstitial lungdisease (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associatedwith rheumatoid arthritis, systemic lupus erythematosus, ankylosingspondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis ordermatomyositis); systemic anaphylaxis or hypersensitivity responses,drug allergies, insect sting allergies; autoimmune diseases, such asrheumatoid arthritis, psoriatic arthritis, multiple sclerosis,Guillain-Barré syndrome, systemic lupus erythematosus, myastenia gravis,glomerulonephritis, autoimmune thyroiditis, graft rejection, includingallograft rejection or graft-versus-host disease; inflammatory boweldiseases, such as Crohn's disease and ulcerative colitis;spondyloarthropathies; scleroderma; psoriasis (including T-cell mediatedpsoriasis) and inflammatory dermatoses such as dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);eosinphilic myotis, and eosiniphilic fasciitis; graft rejection, inparticular but not limited to solid organ transplants, such as heart,lung, liver, kidney, and pancreas transplants (e.g. kidney and lungallografts); epilepsy; pain; fibromyalgia; stroke, e.g., neuroprotectionfollowing a stroke.

In alternative embodiments, each R¹ may be independently H or C₁₋₆ acyl;both R² groups may be joined together with the nitrogen atom to whichthey are attached to form a ring, said ring optionally independentlysubstituted from one up to the maximum number of substituents with oneor more of: fluoro, OH, methyl, or OCH₃; R³ may be OR⁴ or NR⁴ ₂; whereineach R⁴ may be independently selected from the group consisting of: H,C₁₋₆ alkyl, C₃₋₆ cycloalkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆alkynyl, C₁₋₆ acyl, or carbamoyl. The administering may increase thelevel of O-GlcNAc in the subject. The subject may be a human.

In alternative aspects, the invention provides use of a compound of aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof:

where each R¹ may be independently H or C₁₋₆ acyl; both R² groups may bejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ may be OR⁴ or NR⁴ ₂; wherein each R⁴ may beindependently selected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, orcarbamoyl, in the preparation of a medicament. The medicament may be forselectively inhibiting an O-GlcNAcase, for increasing the level ofO-GlcNAc, for treating a condition modulated by an O-GlcNAcase, fortreating a neurodegenerative disease, a tauopathy, a cancer, or stress.

In alternative aspects, the invention provides a method for screeningfor a selective inhibitor of an O-GlcNAcase, by a) contacting a firstsample with a test compound; b) contacting a second sample with acompound of Formula (I)

where each R¹ may be independently H or C₁₋₆ acyl; both R² groups may bejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ may be OR⁴ or NR⁴ ₂; wherein each R⁴ may beindependently selected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, orcarbamoyl; c) determining the level of inhibition of the O-GlcNAcase inthe first and second samples, where the test compound is a selectiveinhibitor of a O-GlcNAcase if the test compound exhibits the same orgreater inhibition of the O-GlcNAcase when compared to the compound ofFormula (I).

This summary of the invention does not necessarily describe all featuresof the invention.

DETAILED DESCRIPTION

The invention provides, in part, novel compounds that are capable ofinhibiting an O-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidase(O-GlcNAcase). In some embodiments, the O-GlcNAcase is a mammalianO-GlcNAcase, such as a rat, mouse or human O-GlcNAcase.

In some embodiments, a compound according to the invention exhibitsselectivity in inhibiting an O-GlcNAcase. In some embodiments, one ormore of the compounds according to the invention are more selective foran O-GlcNAcase over a β-hexosaminidase. In some embodiments, one or moreof the compounds selectively inhibit the activity of a mammalianO-GlcNAcase over a mammalian β-hexosaminidase. In some embodiments, aselective inhibitor of an O-GlcNAcase does not substantially inhibitβ-hexosaminidase. In some embodiments, the β-hexosaminidase is amammalian β-hexosaminidase, such as a rat, mouse or humanβ-hexosaminidase. A compound that “selectively” inhibits an O-GlcNAcaseis a compound that inhibits the activity or biological function of anO-GlcNAcase, but does not substantially inhibit the activity orbiological function of a β-hexosaminidase. For example, in someembodiments, a selective inhibitor of an O-GlcNAcase selectivelyinhibits the cleavage of 2-acetamido-2-deoxy-β-D-glucopyranoside(O-GlcNAc) from polypeptides. In some embodiments, a selective inhibitorof an O-GlcNAcase selectively binds to an O-GlcNAcase. In someembodiments, a selective inhibitor of an O-GlcNAcase inhibitshyperphosphorylation of a tau protein and/or inhibits formations ofNFTs. By “inhibits,” “inhibition” or “inhibiting” means a decrease byany value between 10% and 90%, or of any integer value between 30% and60%, or over 100%, or a decrease by 1-fold, 2-fold, 5-fold, 10-fold ormore. It is to be understood that the inhibiting does not require fullinhibition. In some embodiments, a selective inhibitor of an O-GlcNAcaseelevates or enhances O-GlcNAc levels e.g., O-GlcNAc-modified polypeptideor protein levels, in cells, tissues, or organs (e.g., in brain, muscle,or heart (cardiac) tissue) and in animals. By “elevating” or “enhancing”is meant an increase by any value between 10% and 90%, or of any integervalue between 30% and 60%, or over 100%, or an increase by 1-fold,2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 50-fold, 100-fold or more. Insome embodiments, a selective inhibitor of an O-GlcNAcase exhibits aselectivity ratio, as described herein, in the range 10 to 100000, or inthe range 100 to 100000, or in the range 1000 to 100000, or at least 10,20, 50, 100, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,5000, 6000, 7000, 10,000, 25,000, 50,000, 75,000, or any value within orabout the described range.

One or more of the compounds of the present invention elevate O-GlcNAclevels on O-GlcNAc-modified polypeptides or proteins in vivospecifically via interaction with an O-GlcNAcase enzyme, and areeffective in treating conditions which require or respond to inhibitionof O-GlcNAcase activity.

In some embodiments, one or more of the compounds of the presentinvention are useful as agents that produce a decrease in tauphosphorylation and NFT formation. In some embodiments, one or more ofthe compounds are therefore useful to treat Alzheimer's disease andrelated tauopathies. In some embodiments, one or more of the compoundsare thus capable of treating Alzheimer's disease and related tauopathiesby lowering tau phosphorylation and reducing NFT formation as a resultof increasing tau O-GlcNAc levels. In some embodiments, one or more ofthe compounds produce an increase in levels of O-GlcNAc modification onO-GlcNAc-modified polypeptides or proteins, and are therefore useful fortreatment of disorders responsive to such increases in O-GlcNAcmodification; these disorders include without limitationneurodegenerative, inflammatory, cardiovascular, and immunoregulatorydiseases. In some embodiments, a compound is also useful as a result ofother biological activities related to its ability to inhibit theactivity of glycosidase enzymes. In alternative embodiments, one or moreof the compounds of the invention are valuable tools in studying thephysiological role of O-GlcNAc at the cellular and organismal level.

In alternative embodiments, the invention provides methods of enhancingor elevating levels of protein O-GlcNAc modification in animal subjects,such as, veterinary and human subjects. In alternative embodiments, theinvention provides methods of selectively inhibiting an O-GlcNAcaseenzyme in animal subjects, such as, veterinary and human subjects. Inalternative embodiments, the invention provides methods of inhibitingphosphorylation of tau polypeptides, or inhibiting formation of NFTs, inanimal subjects, such as, veterinary and human subjects.

In specific embodiments, the invention provides compounds describedgenerally by Formula (I) and the salts and prodrug forms thereof:

As set forth in Formula (I): each R¹ may be independently H or C₁₋₆acyl; both R² groups may be joined together with the nitrogen atom towhich they are attached to form a ring, said ring optionallyindependently substituted from one up to the maximum number ofsubstituents with one or more of: fluoro, OH, methyl, or OCH₃; R³ may beOR⁴ or NR⁴ ₂; wherein each R⁴ may be independently selected from thegroup consisting of: H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, C₂₋₆ alkenyl, C₃₋₆cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, or carbamoyl.

In the above Formula (I), each optionally substituted moiety may besubstituted with one or more non-interfering substituents. For example,each optionally substituted moiety may be substituted with one or moreinorganic substituents; phosphoryl; halo; ═O; ═NR⁵; OR; C₁₋₈ alkyl orC₂₋₈ alkenyl optionally containing one or more P, N, O, S, N, F, Cl, Br,I, or B, and optionally substituted with halo; CN; optionallysubstituted carbonyl; NR⁵ ₂; C═NR⁵; an optionally substitutedcarbocyclic or heterocyclic ring; or an optionally substituted aryl orheteroaryl. R⁵ may be H, C₁₋₈ alkyl, C₃₋₆ cycloalkyl, aryl, orheteroaryl.

In some embodiments, R¹ as set forth in Formula (I) may be may be H orC(O)R⁵, where R⁵ may be H, C₁₋₈ alkyl, C₃₋₆ cycloalkyl, aryl, orheteroaryl. In some embodiments, R¹ may be H or C(O)CH₃.

In some embodiments, NR² ₂ as set forth in Formula (I), may beoptionally substituted

where X may be CR⁶ ₂, NR⁶, O, C═O, O(C═O), (C═O)O, NR⁶(C═O), or(C═O)NR⁶; where each R⁶ may be independently H or C₁₋₄ alkyl; and n maybe an integer between 0 and 3. In some embodiments, NR² ₂ may beoptionally substituted 1-aziridinyl, 1-azetidinyl, 1-pyrrolidinyl,1-piperidinyl, 1-morpholino, 1-piperizinyl, azetidin-2-one-1-yl,pyrrolidin-2-one-1-yl, or piperid-2-one-1-yl. In some embodiments, NR² ₂may be

In some embodiments, R³ as set forth in Formula (I) may be either OR⁷ orNR⁷², where each R⁷ may be independently either hydrogen or optionallysubstituted C₁₋₈ alkyl, C₃₋₆ cycloalkyl, C₂₋₈ alkenyl, C₄₋₆cycloalkenyl, C₂₋₈ alkynyl, C₁₋₆ acyl, or carbamoyl. In someembodiments, R³ as set forth in Formula (I) may be OH, NH(cyclopropyl),NH(cyclopentyl), O(CO)NH(CH₂CH₃), O(CO)N(CH₃)₂, O(CO)N(CH₂CH₃)₂, orO(CO)CH₃.

In specific embodiments of the invention, compounds according to Formula(I) include the compounds described in Table 1.

TABLE 1 Example Name Structure  1 (3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol

 2 (3aR,5R,6S,7R,7aR)-5- (acetoxymethyl)-2-(azetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diyl diacetate

 3 (3aR,5R,6S,7R,7aR)-2-(3- fluoroazetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro- 3aH-pyrano[3,2-d]thiazole-6,7-diol

 4 (3aR,5R,6S,7R,7aR)-2-(3- hydroxyazetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro- 3aH-pyrano[3,2-d]thiazole-6,7-diol

 5 (3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(3-methoxyazetidin-1-yl)-5,6,7,7a- tetahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol

 6 (3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(3-methylazetidin-1-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol

 7 (3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol

 8 (3aR,5R,6S,7R,7aR)-5- (acetoxymethyl)-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diyl diacetate

 9 (3aR,5R,6S,7R,7aR)-2-((S)-3- fluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro- 3aH-pyrano[3,2-a]thiazole-6,7-diol

10 (3aR,5R,6S,7R,7aR)-2-((R)-3- fluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro- 3aH-pyrano[3,2-d]thiazole-6,7-diol

11 (3aR,5R,6S,7R,7aR)-2-(3,3- difluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro- 3aH-pyrano[3,2-d]thiazole-6,7-diol

12 (3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(isoxazolidin-2-yl)-5,6,7,7a-tetahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol

13 (3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(piperidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol

14 (3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-morpholino-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol

15 (3aR,5R,6S,7R,7aR)-2-(azetidin-1- yl)-5-((cyclopentylamino)methyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol

16 (3aR,5R,6S,7R,7aR)-2-(azetidin-1- yl)-5-((cyclopropylamino)methyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol

17 ((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyl diethylcarbamate

18 ((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyl dimethylcarbamate

19 ((3aR,5R,6S,7R,7aR)-6,7-dihydroxy- 2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol- 5-yl)methyl dimethylcarbamate

20 ((3aR,5R,6S,7R,7aR)-6,7-dihydroxy- 2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol- 5-yl)methyl diethylcarbamate

21 ((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyl ethylcarbamate

As will be appreciated by a person skilled in the art, Formula (I) abovemay also be represented alternatively as follows:

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. For example, “acompound” refers to one or more of such compounds, while “the enzyme”includes a particular enzyme as well as other family members andequivalents thereof as known to those skilled in the art.

Throughout this application, it is contemplated that the term “compound”or “compounds” refers to the compounds discussed herein and includesprecursors and derivatives of the compounds, including acyl-protectedderivatives, and pharmaceutically acceptable salts of the compounds,precursors, and derivatives. The invention also includes prodrugs of thecompounds, pharmaceutical compositions including the compounds and apharmaceutically acceptable carrier, and pharmaceutical compositionsincluding prodrugs of the compounds and a pharmaceutically acceptablecarrier.

The compounds of the present invention may contain one or moreasymmetric centers and can thus occur as racemates and racemic mixtures,single enantiomers, diastereomeric mixtures and individualdiastereomers. Additional asymmetric centers may be present dependingupon the nature of the various substituents on the molecule. Each suchasymmetric center will independently produce two optical isomers and itis intended that all of the possible optical isomers and diastereomersin mixtures and as pure or partially purified compounds are includedwithin the ambit of this invention. Any formulas, structures or names ofcompounds described in this specification that do not specify aparticular stereochemistry are meant to encompass any and all existingisomers as described above and mixtures thereof in any proportion. Whenstereochemistry is specified, the invention is meant to encompass thatparticular isomer in pure form or as part of a mixture with otherisomers in any proportion.

In general, a “non-interfering substituent” is a substituent whosepresence does not destroy the ability of the compound of Formula (I) tomodulate the activity of the O-GlcNAcase enzyme. Specifically, thepresence of the substituent does not destroy the effectiveness of thecompound as a modulator of the activity of the O-GlcNAcase enzyme.

Suitable non-interfering substituents include: H, alkyl (C₁₋₁₀), alkenyl(C₂₋₁₀), alkynyl (C₂₋₁₀), aryl (5-12 members), arylalkyl, arylalkenyl,or arylalkynyl, each of which may optionally contain one or moreheteroatoms selected from O, S, P, N, F, Cl, Br, I, or B, and each ofwhich may be further substituted, for example, by ═O; or optionallysubstituted forms of acyl, arylacyl, alkyl-alkenyl-, alkynyl- orarylsulfonyl and forms thereof which contain heteroatoms in the alkyl,alkenyl, alkynyl or aryl moieties. Other noninterfering substituentsinclude ═O, ═NR, halo, CN, CF₃, CHF₂, NO₂, OR, SR, NR₂, N₃, COOR, andCONR₂, where R is H or alkyl, cycloalkyl, alkenyl, alkynyl, aryl, orheteroaryl. Where the substituted atom is C, the substituents mayinclude, in addition to the substituents listed above, halo, OOCR,NROCR, where R is H or a substituent set forth above.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, containing nounsaturation and including, for example, from one to ten carbon atoms,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, and which isattached to the rest of the molecule by a single bond. Unless statedotherwise specifically in the specification, the alkyl group may beoptionally substituted by one or more substituents as described herein.Unless stated otherwise specifically herein, it is understood that thesubstitution can occur on any carbon of the alkyl group.

“Alkenyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, containing at least onedouble bond and including, for example, from two to ten carbon atoms,such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, and which isattached to the rest of the molecule by a single bond or a double bond.Unless stated otherwise specifically in the specification, the alkenylgroup may be optionally substituted by one or more substituents asdescribed herein. Unless stated otherwise specifically herein, it isunderstood that the substitution can occur on any carbon of the alkenylgroup.

“Alkynyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, containing at least onetriple bond and including, for example, from two to ten carbon atoms.Unless stated otherwise specifically in the specification, the alkenylgroup may be optionally substituted by one or more substituents asdescribed herein.

“Aryl” refers to a phenyl or naphthyl group, including for example, 5-12members. Unless stated otherwise specifically herein, the term “aryl” ismeant to include aryl groups optionally substituted by one or moresubstituents as described herein.

“Heteroaryl” refers to a single or fused aromatic ring group containingone or more heteroatoms in the ring, for example N, O, S, including forexample, 5-14 members. Examples of heteroaryl groups include furan,thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole,isothiazole, 1,2,3-oxadiazole, 1,2,3-triazole, 1,2,4-triazole,1,3,4-thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine,pyrazine, 1,3,5-triazine, imidazole, benzimidazole, benzoxazole,benzothiazole, indolizine, indole, isoindole, benzofuran,benzothiophene, 1H-indazole, purine, 4H-quinolizine, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,1,8-naphthyridine, pteridine. Unless stated otherwise specificallyherein, the term “heteroaryl” is meant to include heteroaryl groupsoptionally substituted by one or more substituents as described herein.

“Arylalkyl” refers to a group of the formula —R_(a)R_(b) where R_(a) isa C₁₋₁₀ alkyl group as described herein and R_(b) is one or more arylmoieties as described herein. The aryl group(s) and the alkyl group maybe optionally substituted as described herein.

“Arylalkenyl” refers to a group of the formula —R_(c)R_(b) where R_(c)is an alkenyl moiety as described herein and R_(b) is one or more arylgroups as described herein. The aryl group(s) and the alkenyl group maybe optionally substituted as described herein.

“Arylalkynyl” refers to a group of the formula —R_(d)R_(b) where R_(d)is an alkynyl moiety as described herein and R_(b) is one or more arylgroups as described herein. The aryl group(s) and the alkynyl group maybe optionally substituted as described herein.

“Acyl” refers to a group of the formula —C(O)R_(a), where R_(a) is aC₁₋₁₀ alkyl group as described herein. The alkyl group may be optionallysubstituted as described herein.

“Arylacyl” refers to a group of the formula —C(O)R_(b), where R_(b) isan aryl or heteroaryl group as described herein. The aryl or heteroarylgroup(s) may be optionally substituted as described herein.

“Carbamoyl” refers to a group of the formula —C(O)N(R_(e))₂, where eachR_(e) is independently H or a C₁₋₁₀ alkyl or C₃₋₁₅ cycloalkyl group asdescribed herein. The alkyl or cycloalkyl group(s) may be optionallysubstituted as described herein.

“Alkoxy” refers to a group of the formula —OR_(a), where R_(a) is aC₁₋₁₀ alkyl group as described herein. The alkyl group(s) may beoptionally substituted as described herein.

“Cycloalkyl” refers to a stable monovalent monocyclic, bicyclic ortricyclic hydrocarbon group consisting solely of carbon and hydrogenatoms, having for example from 3 to 15 carbon atoms, and which issaturated and attached to the rest of the molecule by a single bond.Unless otherwise stated specifically herein, the term “cycloalkyl” ismeant to include cycloalkyl groups which are optionally substituted asdescribed herein.

“Cycloalkenyl” refers to a stable monovalent monocyclic, bicyclic ortricyclic hydrocarbon group consisting solely of carbon and hydrogenatoms, containing at least one double bond, having for example from 3 to15 carbon atoms, and which is attached to the rest of the molecule by asingle bond. Unless otherwise stated specifically herein, the term“cycloalkenyl” is meant to include cycloalkenyl groups which areoptionally substituted as described herein.

In some embodiments, two R² groups as set forth in Formula (I) may beconnected together with the nitrogen atom to which they are attached toform a ring. In these embodiments, “ring” refers to a stablenitrogen-containing monocyclic group having 3 to 6 members that may besaturated or monounsaturated. In alternative embodiments, the ring mayinclude C, H and N atoms. In other embodiments, the ring may includeheteroatoms, for example O and S. Examples of a ring in theseembodiments include 1-aziridinyl, 1-azetidinyl, 1-pyrrolidinyl,2,5-dihydro-1H-pyrrol-1-yl, 1-piperidinyl,1,2,3,6-tetrahydropyridin-1-yl, morpholin-4-yl, thiomorpholin-4-yl,1-piperizinyl, azetidin-2-one-1-yl, pyrrolidin-2-one-1-yl,piperid-2-one-1-yl, 1,2-oxazetidin-2-yl, isoxazolidin-2-yl, and1,2-oxazinan-2-yl. The ring in these embodiments may be optionallysubstituted as described herein.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs one or more times andinstances in which it does not. For example, “optionally substitutedalkyl” means that the alkyl group may or may not be substituted, andthat the description includes both substituted alkyl groups and alkylgroups having no substitution, and that said alkyl groups may besubstituted one or more times. Examples of optionally substituted alkylgroups include, without limitation, methyl, ethyl, propyl, etc. andincluding cycloalkyls such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, etc.; examples of optionally substitutedalkenyl groups include allyl, crotyl, 2-pentenyl, 3-hexenyl,2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl,2-cyclohexenylmethyl, etc. In some embodiments, optionally substitutedalkyl and alkenyl groups include C₁₋₆ alkyls or alkenyls.

“Halo” refers to bromo, chloro, fluoro, iodo, etc. In some embodiments,suitable halogens include fluorine or chlorine.

Therapeutic Indications

The invention provides methods of treating conditions that aremodulated, directly or indirectly, by an O-GlcNAcase enzyme or byO-GlcNAc-modified protein levels, for example, a condition that isbenefited by inhibition of an O-GlcNAcase enzyme or by an elevation ofO-GlcNAc-modified protein levels. Such conditions include, withoutlimitation, Glaucoma, Schizophrenia, tauopathies, such as Alzheimer'sdisease, neurodegenerative diseases, cardiovascular diseases, diseasesassociated with inflammation, diseases associated with immunosuppressionand cancers. One or more of the compounds of the invention are alsouseful in the treatment of diseases or disorders related to deficiencyor over-expression of O-GlcNAcase or accumulation or depletion ofO-GlcNAc, or any disease or disorder responsive to glycosidaseinhibition therapy. Such diseases and disorders include, but are notlimited to, Glaucoma, Schizophrenia, neurodegenerative disorders, suchas Alzheimer's disease (AD), or cancer. Such diseases and disorders mayalso include diseases or disorders related to the accumulation ordeficiency in the enzyme OGT. Also included is a method of protecting ortreating target cells expressing proteins that are modified by O-GlcNAcresidues, the dysregulation of which modification results in disease orpathology. The term “treating” as used herein includes treatment,prevention, and amelioration.

In alternative embodiments, the invention provides methods of enhancingor elevating levels of protein O-GlcNAc modification in animal subjects,such as, veterinary and human subjects. This elevation of O-GlcNAclevels can be useful for the prevention or treatment of Alzheimer'sdisease; prevention or treatment of other neurodegenerative diseases(e.g. Parkinson's disease, Huntington's disease); providingneuroprotective effects; preventing damage to cardiac tissue; andtreating diseases associated with inflammation or immunosuppression.

In alternative embodiments, the invention provides methods ofselectively inhibiting an O-GlcNAcase enzyme in animal subjects, such asveterinary and human subjects.

In alternative embodiments, the invention provides methods of inhibitingphosphorylation of tau polypeptides, or inhibiting formation of NFTs, inanimal subjects, such as, veterinary and human subjects. Accordingly, acompound of the invention may be used to study and treat AD and othertauopathies.

In general, the methods of the invention are effected by administering acompound according to the invention to a subject in need thereof, or bycontacting a cell or a sample with a compound according to theinvention, for example, a pharmaceutical composition comprising atherapeutically effective amount of the compound according to Formula(I). More particularly, they are useful in the treatment of a disorderin which the regulation of O-GlcNAc protein modification is implicated,or any condition as described herein. Disease states of interest includeAlzheimer's disease (AD) and related neurodegenerative tauopathies, inwhich abnormal hyperphosphorylation of the microtubule-associatedprotein tau is involved in disease pathogenesis. In some embodiments, acompound may be used to block hyperphosphorylation of tau by maintainingelevated levels of O-GlcNAc on tau, thereby providing therapeuticbenefit.

The effectiveness of a compound in treating pathology associated withthe accumulation of toxic tau species (for example, Alzheimer's diseaseand other tauopathies) may be confirmed by testing the ability of acompound to block the formation of toxic tau species in establishedcellular¹¹⁸⁻¹²⁰ and/or transgenic animal models of disease.^(32,33)

Tauopathies that may be treated with a compound of the inventioninclude: Alzheimer's disease, Amyotrophic lateral sclerosis (ALS),Amyotrophic lateral sclerosis with cognitive impairment (ALSci),Argyrophilic grain dementia, Bluit disease, Corticobasal degeneration(CBD), Dementia pugilistica, Diffuse neurofibrillary tangles withcalcification, Down's syndrome, Familial British dementia, FamilialDanish dementia, Frontotemporal dementia with parkinsonism linked tochromosome 17 (FTDP-17), Gerstmann-Straussler-Scheinker disease,Guadeloupean parkinsonism, Hallevorden-Spatz disease (neurodegenerationwith brain iron accumulation type 1), Multiple system atrophy, Myotonicdystrophy, Niemann-Pick disease (type C), Pallido-ponto-nigraldegeneration, Parkinsonism-dementia complex of Guam, Pick's disease(PiD), Post-encephalitic parkinsonism (PEP), Prion diseases (includingCreutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Fatal Familial Insomnia, and Kuru), Progressive supercorticalgliosis, Progressive supranuclear palsy (PSP), Richardson's syndrome,Subacute sclerosing panencephalitis, Tangle-only dementia, and Glaucoma.

One or more of the compounds of this invention are also useful in thetreatment of conditions associate with tissue damage or stress,stimulating cells, or promoting differentiation of cells. Accordingly,in some embodiments, a compound of this invention may be used to providetherapeutic benefit in a variety of conditions or medical proceduresinvolving stress in cardiac tissue, including but not limited to:ischemia; hemorrhage; hypovolemic shock; myocardial infarction; aninterventional cardiology procedure; cardiac bypass surgery;fibrinolytic therapy; angioplasty; and stent placement.

The effectiveness of a compound in treating pathology associated withcellular stress (including ischemia, hemorrhage, hypovolemic shock,myocardial infarction, and other cardiovascular disorders) may beconfirmed by testing the ability of a compound to prevent cellulardamage in established cellular stress assays,^(105,116,117) and toprevent tissue damage and promote functional recovery in animal modelsof ischemia-reperfusion,^(70,114) and trauma-hemorrhage.^(72,112,115)

Compounds that selectively inhibit O-GlcNAcase activity may be used forthe treatment of diseases that are associated with inflammation,including but not limited to, inflammatory or allergic diseases such asasthma, allergic rhinitis, hypersensitivity lung diseases,hypersensitivity pneumonitis, eosinophilic pneumonias, delayed-typehypersensitivity, atherosclerosis, interstitial lung disease (ILD)(e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoidarthritis, systemic lupus erythematosus, ankylosing spondylitis,systemic sclerosis, Sjogren's syndrome, polymyositis ordermatomyositis); systemic anaphylaxis or hypersensitivity responses,drug allergies, insect sting allergies; autoimmune diseases, such asrheumatoid arthritis, psoriatic arthritis, multiple sclerosis,Guillain-Barré syndrome, systemic lupus erythematosus, myastenia gravis,glomerulonephritis, autoimmune thyroiditis, graft rejection, includingallograft rejection or graft-versus-host disease; inflammatory boweldiseases, such as Crohn's disease and ulcerative colitis;spondyloarthropathies; scleroderma; psoriasis (including T-cell mediatedpsoriasis) and inflammatory dermatoses such as dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);eosinphilic myotis, eosiniphilic fasciitis; and cancers.

In addition, compounds that affects levels of protein O-GlcNAcmodification may be used for the treatment of diseases associated withimmunosuppression, such as in individuals undergoing chemotherapy,radiation therapy, enhanced wound healing and burn treatment, therapyfor autoimmune disease or other drug therapy (e.g., corticosteroidtherapy) or combination of conventional drugs used in the treatment ofautoimmune diseases and graft/transplantation rejection, which causesimmunosuppression; or immunosuppression due to congenital deficiency inreceptor function or other causes.

One or more of the compounds of the invention may be useful fortreatment of neurodegenerative diseases, including Parkinson's diseaseand Huntington's disease. Other conditions that may be treated are thosetriggered, affected, or in any other way correlated with levels ofO-GlcNAc post-translational protein modification. It is expected thatone or more of the compounds of this invention may be useful for thetreatment of such conditions and in particular, but not limited to, thefollowing for which a association with O-GlcNAc levels on proteins hasbeen established: graft rejection, in particular but not limited tosolid organ transplants, such as heart, lung, liver, kidney, andpancreas transplants (e.g. kidney and lung allografts); cancer, inparticular but not limited to cancer of the breast, lung, prostate,pancreas, colon, rectum, bladder, kidney, ovary; as well asnon-Hodgkin's lymphoma and melanoma; epilepsy, pain, fibromyalgia, orstroke, e.g., for neuroprotection following a stroke.

Pharmaceutical & Veterinary Compositions, Dosages, and Administration

Pharmaceutical compositions including compounds according to theinvention, or for use according to the invention, are contemplated asbeing within the scope of the invention. In some embodiments,pharmaceutical compositions including an effective amount of a compoundof Formula (I) are provided.

The compounds of Formula (I) and their pharmaceutically acceptablesalts, enantiomers, solvates, and derivatives are useful because theyhave pharmacological activity in animals, including humans. In someembodiments, one or more of the compounds according to the invention arestable in plasma, when administered to a subject.

In some embodiments, a compound according to the invention, or for useaccording to the invention, may be provided in combination with anyother active agents or pharmaceutical compositions where such combinedtherapy is useful to modulate O-GlcNAcase activity, for example, totreat neurodegenerative, inflammatory, cardiovascular, orimmunoregulatory diseases, or any condition described herein. In someembodiments, a compound according to the invention, or for use accordingto the invention, may be provided in combination with one or more agentsuseful in the prevention or treatment of Alzheimer's disease. Examplesof such agents include, without limitation,

-   -   acetylcholine esterase inhibitors (AChEIs) such as Aricept®        (Donepezil), Exelon® (Rivastigmine), Razadyne® (Razadyne ER®,        Reminyl®, Nivalin®, Galantamine), Cognex® (Tacrine), Dimebon,        Huperzine A, Phenserine, Debio-9902 SR (ZT-1 SR), Zanapezil        (TAK0147), ganstigmine, NP7557, etc.;    -   NMDA receptor antagonists such as Namenda® (Axura®, Akatinol®,        Ebixa®, Memantine), Dimebon, SGS-742, Neramexane, Debio-9902 SR        (ZT-1 SR), etc.;    -   gamma-secretase inhibitors and/or modulators such as Flurizan™        (Tarenflurbil, MPC-7869, R-flurbiprofen), LY450139, MK 0752,        E2101, BMS-289948, BMS-299897, BMS-433796, LY-411575, GSI-136,        etc.;    -   beta-secretase inhibitors such as ATG-Z1, CTS-21166, etc.;    -   alpha-secretase activators, such as NGX267, etc;    -   amyloid-β aggregation and/or fibrillization inhibitors such as        Alzhemed™ (3APS, Tramiprosate, 3-amino-1-propanesulfonic acid),        AL-108, AL-208, AZD-103, PBT2, Cereact, ONO-2506PO, PPI-558,        etc.;    -   tau aggregation inhibitors such as methylene blue, etc.;    -   microtubule stabilizers such as AL-108, AL-208, paclitaxel,        etc.;    -   RAGE inhibitors, such as TTP488, etc.;    -   5-HT1a receptor antagonists, such as Xaliproden, Lecozotan,        etc.;    -   5-HT4 receptor antagonists, such as PRX-03410, etc.;

kinase inhibitors such as SRN-003-556, amfurindamide, LiCl, AZD1080,NP031112, SAR-502250, etc.

-   -   humanized monoclonal anti-Aβ antibodies such as Bapineuzumab        (AAB-001), LY2062430, RN1219, ACU-5A5, etc.;    -   amyloid vaccines such as AN-1792, ACC-001    -   neuroprotective agents such as Cerebrolysin, AL-108, AL-208,        Huperzine A, etc.;    -   L-type calcium channel antagonists such as MEM-1003, etc.;    -   nicotinic receptor antagonists, such as AZD3480, GTS-21, etc.;    -   nicotinic receptor agonists, such as MEM 3454, Nefiracetam,        etc.;    -   peroxisome proliferator-activated receptor (PPAR) gamma agonists        such as Avandia® (Rosglitazone), etc.;    -   phosphodiesterase IV (PDE4) inhibitors, such as MK-0952, etc.;    -   hormone replacement therapy such as estrogen (Premarin), etc.;    -   monoamine oxidase (MAO) inhibitors such as NS2330, Rasagiline        (Azilect®), TVP-1012, etc.;    -   AMPA receptor modulators such as Ampalex (CX 516), etc.;    -   nerve growth factors or NGF potentiators, such as CERE-110        (AAV-NGF), T-588, T-817MA, etc.;    -   agents that prevent the release of luteinizing hormone (LH) by        the pituitary gland, such as leuoprolide (VP-4896), etc.;    -   GABA receptor modulators such as AC-3933, NGD 97-1, CP-457920,        etc.;    -   benzodiazepine receptor inverse agonists such as SB-737552        (S-8510), AC-3933, etc.;    -   noradrenaline-releasing agents such as T-588, T-817MA, etc.

It is to be understood that combination of compounds according to theinvention, or for use according to the invention, with Alzheimer'sagents is not limited to the examples described herein, but includescombination with any agent useful for the treatment of Alzheimer'sdisease. Combination of compounds according to the invention, or for useaccording to the invention, and other Alzheimer's agents may beadministered separately or in conjunction. The administration of oneagent may be prior to, concurrent to, or subsequent to theadministration of other agent(s).

In alternative embodiments, a compound may be supplied as a “prodrug” orprotected forms, which release the compound after administration to asubject. For example, a compound may carry a protective group which issplit off by hydrolysis in body fluids, e.g., in the bloodstream, thusreleasing the active compound or is oxidized or reduced in body fluidsto release the compound. Accordingly, a “prodrug” is meant to indicate acompound that may be converted under physiological conditions or bysolvolysis to a biologically active compound of the invention. Thus, theterm “prodrug” refers to a metabolic precursor of a compound of theinvention that is pharmaceutically acceptable. A prodrug may be inactivewhen administered to a subject in need thereof, but is converted in vivoto an active compound of the invention. Prodrugs are typically rapidlytransformed in vivo to yield the parent compound of the invention, forexample, by hydrolysis in blood. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in asubject.

The term “prodrug” is also meant to include any covalently bondedcarriers which release the active compound of the invention in vivo whensuch prodrug is administered to a subject. Prodrugs of a compound of theinvention may be prepared by modifying functional groups present in thecompound of the invention in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to the parentcompound of the invention. Prodrugs include compounds of the inventionwherein a hydroxy, amino or mercapto group is bonded to any group that,when the prodrug of the compound of the invention is administered to amammalian subject, cleaves to form a free hydroxy, free amino or freemercapto group, respectively. Examples of prodrugs include, but are notlimited to, acetate, formate and benzoate derivatives of alcohol andacetamide, formamide, and benzamide derivatives of amine functionalgroups in one or more of the compounds of the invention and the like.

A discussion of prodrugs may be found in “Smith and Williams'Introduction to the Principles of Drug Design,” H. J. Smith, Wright,Second Edition, London (1988); Bundgard, H., Design of Prodrugs (1985),pp. 7-9, 21-24 (Elsevier, Amsterdam); The Practice of MedicinalChemistry, Camille G. Wermuth et al., Ch 31, (Academic Press, 1996); ATextbook of Drug Design and Development, P. Krogsgaard-Larson and H.Bundgaard, eds. Ch 5, pgs 113 191 (Harwood Academic Publishers, 1991);Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S.Symposium Series, Vol. 14; or in Bioreversible Carriers in Drug Design,ed. Edward B. Roche, American Pharmaceutical Association and PergamonPress, 1987, all of which are incorporated in full by reference herein.

Suitable prodrug forms of one or more of the compounds of the inventioninclude embodiments in which R¹ is C(O)R or R³ is O(CO)R, where R isoptionally substituted alkyl, alkenyl, alkynyl, aryl, or heteroaryl. Inthese cases the ester groups may be hydrolyzed in vivo (e.g. in bodilyfluids), releasing the active compounds in which R¹ is H and R³ is OH.Preferred prodrug embodiments of the invention include compounds ofFormula (I) where one or two of R¹ is C(O)CH₃. Preferred prodrugembodiments of the invention also include compounds of Formula (I) whereR³ is O(CO)CH₃.

Compounds according to the invention, or for use according to theinvention, can be provided alone or in combination with other compoundsin the presence of a liposome, an adjuvant, or any pharmaceuticallyacceptable carrier, diluent or excipient, in a form suitable foradministration to a subject such as a mammal, for example, humans,cattle, sheep, etc. If desired, treatment with a compound according tothe invention may be combined with more traditional and existingtherapies for the therapeutic indications described herein. Compoundsaccording to the invention may be provided chronically orintermittently. “Chronic” administration refers to administration of thecompound(s) in a continuous mode as opposed to an acute mode, so as tomaintain the initial therapeutic effect (activity) for an extendedperiod of time. “Intermittent” administration is treatment that is notconsecutively done without interruption, but rather is cyclic in nature.The terms “administration,” “administrable,” or “administering” as usedherein should be understood to mean providing a compound of theinvention to the subject in need of treatment.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier that has been approved, for example, bythe United States Food and Drug Administration or other governmentalagency as being acceptable for use in humans or domestic animals.

A compound of the present invention may be administered in the form of apharmaceutically acceptable salt. In such cases, pharmaceuticalcompositions in accordance with this invention may comprise a salt ofsuch a compound, preferably a physiologically acceptable salt, which areknown in the art. In some embodiments, the term “pharmaceuticallyacceptable salt” as used herein means an active ingredient comprisingcompounds of Formula I used in the form of a salt thereof, particularlywhere the salt form confers on the active ingredient improvedpharmacokinetic properties as compared to the free form of the activeingredient or other previously disclosed salt form.

A “pharmaceutically acceptable salt” includes both acid and baseaddition salts. A “pharmaceutically acceptable acid addition salt”refers to those salts which retain the biological effectiveness andproperties of the free bases, which are not biologically or otherwiseundesirable, and which are formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like.

A “pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline and caffeine.

Thus, the term “pharmaceutically acceptable salt” encompasses allacceptable salts including but not limited to acetate, lactobionate,benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate,bisulfate, mandelate, bitartarate, mesylate, borate, methylbromide,bromide, methylnitrite, calcium edetate, methylsulfate, camsylate,mucate, carbonate, napsylate, chloride, nitrate, clavulanate,N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate,edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate,esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate,polygalacturonate, gluconate, salicylate, glutame, stearate,glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydradamine,succinate, hydrobromide, tannate, hydrochloride, tartrate,hydroxynaphthoate, teoclate, iodide, tosylate, isothionate,triethiodide, lactate, panoate, valerate, and the like.

Pharmaceutically acceptable salts of a compound of the present inventioncan be used as a dosage for modifying solubility or hydrolysischaracteristics, or can be used in sustained release or prodrugformulations. Also, pharmaceutically acceptable salts of a compound ofthis invention may include those formed from cations such as sodium,potassium, aluminum, calcium, lithium, magnesium, zinc, and from basessuch as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylene-diamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethyl-amine, diethylamine,piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammoniumhydroxide.

Pharmaceutical formulations will typically include one or more carriersacceptable for the mode of administration of the preparation, be it byinjection, inhalation, topical administration, lavage, or other modessuitable for the selected treatment. Suitable carriers are those knownin the art for use in such modes of administration.

Suitable pharmaceutical compositions may be formulated by means known inthe art and their mode of administration and dose determined by theskilled practitioner. For parenteral administration, a compound may bedissolved in sterile water or saline or a pharmaceutically acceptablevehicle used for administration of non-water soluble compounds such asthose used for vitamin K. For enteral administration, the compound maybe administered in a tablet, capsule or dissolved in liquid form. Thetable or capsule may be enteric coated, or in a formulation forsustained release. Many suitable formulations are known, including,polymeric or protein microparticles encapsulating a compound to bereleased, ointments, gels, hydrogels, or solutions which can be usedtopically or locally to administer a compound. A sustained release patchor implant may be employed to provide release over a prolonged period oftime. Many techniques known to skilled practitioners are described inRemington: the Science & Practice of Pharmacy by Alfonso Gennaro,20^(th) ed., Williams & Wilkins, (2000). Formulations for parenteraladministration may, for example, contain excipients, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin, orhydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of a compound. Otherpotentially useful parenteral delivery systems for modulatory compoundsinclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationmay contain excipients, for example, lactose, or may be aqueoussolutions containing, for example, polyoxyethylene-9-lauryl ether,glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

A compound or a pharmaceutical composition according to the presentinvention may be administered by oral or non-oral, e.g., intramuscular,intraperitoneal, intravenous, intracisternal injection or infusion,subcutaneous injection, transdermal or transmucosal routes. In someembodiments, a compound or pharmaceutical composition in accordance withthis invention or for use in this invention may be administered by meansof a medical device or appliance such as an implant, graft, prosthesis,stent, etc. Implants may be devised which are intended to contain andrelease such compounds or compositions. An example would be an implantmade of a polymeric material adapted to release the compound over aperiod of time. A compound may be administered alone or as a mixturewith a pharmaceutically acceptable carrier e.g., as solid formulationssuch as tablets, capsules, granules, powders, etc.; liquid formulationssuch as syrups, injections, etc.; injections, drops, suppositories,pessaryies. In some embodiments, compounds or pharmaceuticalcompositions in accordance with this invention or for use in thisinvention may be administered by inhalation spray, nasal, vaginal,rectal, sublingual, or topical routes and may be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and vehiclesappropriate for each route of administration.

A compound of the invention may be used to treat animals, includingmice, rats, horses, cattle, sheep, dogs, cats, and monkeys. However, acompound of the invention can also be used in other organisms, such asavian species (e.g., chickens). One or more of the compounds of theinvention may also be effective for use in humans. The term “subject” oralternatively referred to herein as “patient” is intended to be referredto an animal, preferably a mammal, most preferably a human, who has beenthe object of treatment, observation or experiment. However, one or moreof the compounds, methods and pharmaceutical compositions of the presentinvention may be used in the treatment of animals. Accordingly, as usedherein, a “subject” may be a human, non-human primate, rat, mouse, cow,horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected ofhaving or at risk for having a condition requiring modulation ofO-GlcNAcase activity.

An “effective amount” of a compound according to the invention includesa therapeutically effective amount or a prophylactically effectiveamount. A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result, such as inhibition of an O-GlcNAcase,elevation of O-GlcNAc levels, inhibition of tau phosphorylation, or anycondition described herein. A therapeutically effective amount of acompound may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the compound toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the compound are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result, such as inhibition of an O-GlcNAcase,elevation of O-GlcNAc levels, inhibition of tau phosphorylation, or anycondition described herein. Typically, a prophylactic dose is used insubjects prior to or at an earlier stage of disease, so that aprophylactically effective amount may be less than a therapeuticallyeffective amount. A suitable range for therapeutically orprophylactically effective amounts of a compound may be any integer from0.1 nM-0.1 M, 0.1 nM-0.05 M, 0.05 nM-15 μM or 0.01 nM-10 μM.

In alternative embodiments, in the treatment or prevention of conditionswhich require modulation of O-GlcNAcase activity, an appropriate dosagelevel will generally be about 0.01 to 500 mg per kg subject body weightper day, and can be administered in singe or multiple doses. In someembodiments, the dosage level will be about 0.1 to about 250 mg/kg perday. It will be understood that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific compound used,the metabolic stability and length of action of that compound, the age,body weight, general health, sex, diet, mode and time of administration,rate of excretion, drug combination, the severity of the particularcondition, and the patient undergoing therapy.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. For any particular subject, specific dosageregimens may be adjusted over time according to the individual need andthe professional judgement of the person administering or supervisingthe administration of the compositions. Dosage ranges set forth hereinare exemplary only and do not limit the dosage ranges that may beselected by medical practitioners. The amount of active compound(s) inthe composition may vary according to factors such as the disease state,age, sex, and weight of the subject. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, a single bolusmay be administered, several divided doses may be administered over timeor the dose may be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. It may be advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. In general, compounds of theinvention should be used without causing substantial toxicity, and asdescribed herein, one or more of the compounds exhibit a suitable safetyprofile for therapeutic use. Toxicity of a compound of the invention canbe determined using standard techniques, for example, by testing in cellcultures or experimental animals and determining the therapeutic index,i.e., the ratio between the LD50 (the dose lethal to 50% of thepopulation) and the LD100 (the dose lethal to 100% of the population).In some circumstances however, such as in severe disease conditions, itmay be necessary to administer substantial excesses of the compositions.

In the compounds of generic Formula (I), the atoms may exhibit theirnatural isotopic abundances, or one or more of the atoms may beartificially enriched in a particular isotope having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number predominantly found in nature. The present invention ismeant to include all suitable isotopic variations of the compounds ofgeneric Formula (I). For example, different isotopic forms of hydrogen(H) include protium (¹H), deuterium (²H) and tritium (³H). Protium isthe predominant hydrogen isotope found in nature. Enriching fordeuterium may afford certain therapeutic advantages, such as increasingin vivo half-life or reducing dosage requirements, or may provide acompound useful as a standard for characterization of biologicalsamples. Isotopically-enriched compounds within generic Formula (I) canbe prepared without undue experimentation by conventional techniqueswell known to those skilled in the art or by processes analogous tothose described in the Schemes and Examples herein using appropriateisotopically-enriched reagents and/or intermediates.

Other Uses and Assays

A compound of Formula (I) may be used in screening assays for compoundswhich modulate the activity of glycosidase enzymes, preferably theO-GlcNAcase enzyme. The ability of a test compound to inhibitO-GlcNAcase-dependent cleavage of O-GlcNAc from a model substrate may bemeasured using any assays, as described herein or known to one ofordinary skill in the art. For example, a fluoresence or UV-based assayknown in the art may be used. A “test compound” is anynaturally-occurring or artificially-derived chemical compound. Testcompounds may include, without limitation, peptides, polypeptides,synthesised organic molecules, naturally occurring organic molecules,and nucleic acid molecules. A test compound can “compete” with a knowncompound such as a compound of Formula (I) by, for example, interferingwith inhibition of O-GlcNAcase-dependent cleavage of O-GlcNAc or byinterfering with any biological response induced by a compound ofFormula (I).

Generally, a test compound can exhibit any value between 10% and 200%,or over 500%, modulation when compared to a compound of Formula (I) orother reference compound. For example, a test compound may exhibit atleast any positive or negative integer from 10% to 200% modulation, orat least any positive or negative integer from 30% to 150% modulation,or at least any positive or negative integer from 60% to 100%modulation, or any positive or negative integer over 100% modulation. Acompound that is a negative modulator will in general decreasemodulation relative to a known compound, while a compound that is apositive modulator will in general increase modulation relative to aknown compound.

In general, test compounds are identified from large libraries of bothnatural products or synthetic (or semi-synthetic) extracts or chemicallibraries according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the method(s) ofthe invention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited to,plant-, fungal-, prokaryotic- or animal-based extracts, fermentationbroths, and synthetic compounds, as well as modification of existingcompounds. Numerous methods are also available for generating random ordirected synthesis (e.g., semi-synthesis or total synthesis) of anynumber of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts are commercially available from a number of sources,including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor BranchOceanographic Institute (Ft. Pierce, Fla., USA), and PharmaMar, MA, USA.In addition, natural and synthetically produced libraries are produced,if desired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

When a crude extract is found to modulate inhibition ofO-GlcNAcase-dependent cleavage of O-GlcNAc, or any biological responseinduced by a compound of Formula (I), further fractionation of thepositive lead extract is necessary to isolate chemical constituentsresponsible for the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract havingO-GlcNAcase-inhibitory activities. The same assays described herein forthe detection of activities in mixtures of compounds can be used topurify the active component and to test derivatives thereof. Methods offractionation and purification of such heterogeneous extracts are knownin the art. If desired, compounds shown to be useful agents fortreatment are chemically modified according to methods known in the art.Compounds identified as being of therapeutic, prophylactic, diagnostic,or other value may be subsequently analyzed using a suitable animalmodel, as described herein on known in the art.

In some embodiments, one or more of the compounds are useful in thedevelopment of animal models for studying diseases or disorders relatedto deficiencies in O-GlcNAcase, over-expression of O-GlcNAcase,accumulation of O-GlcNAc, depletion of O-GlcNAc, and for studyingtreatment of diseases and disorders related to deficiency orover-expression of O-GlcNAcase, or accumulation or depletion ofO-GlcNAc. Such diseases and disorders include neurodegenerativediseases, including Alzheimer's disease, and cancer.

Various alternative embodiments and examples of the invention aredescribed herein. These embodiments and examples are illustrative andshould not be construed as limiting the scope of the invention.

EXAMPLES

The following examples are intended to illustrate embodiments of theinvention and are not intended to be construed in a limiting manner.

Examples 1 & 2(3aR,5R,6S,7R,7aR)-5-(acetoxymethyl)-2-(azetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diyldiacetate (2) and(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol(3)

(3aR,5R,6S,7R,7aR)-5-(acetoxymethyl)-2-(azetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diyldiacetate (2)

A solution of azetidine hydrochloride (12 g, 129 mmol) and(3R,4R,5S,6R)-6-(acetoxymethyl)-3-isothiocyanato-tetrahydro-2H-pyran-2,4,5-triyltriacetate (48 g, 123 mmol) in dichloromethane (500 mL) was treated withtriethylamine (18.7 g, 185 mmol) for 1 h at room temperature, andfollowed by addition oftrifluoroacetic acid (56.2 g, 493 mmol). Thereaction mixture was stirred overnight at room temperature, and thenquenched by aqueous sodium bicarbonate. The organic layer was separated,dried over anhydrous MgSO₄, and condensed under vacuum to give aresidue, which was purified by a silica gel column, eluted with 1%methanol in dichloromethane to provide compound 2 as a light yellowsyrup (29 g, 61%). (ES, m/z) [M+H]⁺ 386.9; ¹H NMR (300 MHz, CDCl₃)6.28-6.30 (d, J=6.6 Hz, 1H), 5.44-5.46 (m, 1H), 4.95-4.99 (m, 1H),4.34-4.37 (t, J=5.4 Hz, 1H), 4.04-4.17 (m, 6H), 3.86-3.92 (m, 1H),2.34-2.44 (m, 2H), 2.06-2.14 (m, 9H).

Step 2

(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol(3)

A solution of(3aR,5R,6S,7R,7aR)-5-(acetoxymethyl)-2-(azetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diyldiacetate (360 mg, 0.93 mmol) in methanol (5 mL) was treated withpotassium carbonate (13 mg, 0.09 mmol) overnight at 40° C. The reactionmixture was concentrated under vacuum to give a residue, which waspurified by Prep-HPLC with the following conditions [(Agilent 1200 prepHPLC): Column, X-Bridge C18, 19*50 mm 3.5 um; mobile phase, Water with0.05% NH₄OH and CH₃CN (3% CH₃CN up to 9% in 5 min; Detector, UV 220 nm)]to give compound 3 as a white solid (100 mg, 40%). (ES, m/z) [M+H]⁺261.0; ¹H NMR (300 MHz, DMSO+D₂O) δ 6.25-6.27 (d, J=6.3 Hz, 1H),3.92-3.96 (t, J=5.7 Hz, 1H), 3.83-3.88 (m, 4H), 3.68-3.71 (t, J=4.8 Hz,1H), 3.55-3.58 (m, 1H), 3.25-3.42 (m, 3H), 2.21-2.26 (m, 2H).

The following examples were synthesized according to proceduresanalogous to the schemes and examples outlined above.

TABLE 2 Example Structure Name MH+  3

(3aR,5R,6S,7R,7aR)-2-(3- fluoroazetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol279.0  4

(3aR,5R,6S,7R,7aR)-2-(3- hydroxyazetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol276.9  5

(3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(3-methoxyazetidin-1-yl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol 291.0  6

(3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(3-methylazetidin-1-yl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol 275.0  7

(3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol 275.0  8

(3aR,5R,6S,7R,7aR)-5- (acetoxymethyl)-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diyl diacetate400.9  9

(3aR,5R,6S,7R,7aR)-2-((S)-3- fluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol293.0 10

(3aR,5R,6S,7R,7aR)-2-((R)-3- fluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol293.0 11

(3aR,5R,6S,7R,7aR)-2-(3,3- difluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a- tetrahydro-3aH-pyrano[3,2- d]thiazole-6,7-diol311.0 12

(3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(isoxazolidin-2-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol 277.0 13

(3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-(piperidin-1-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol 288.9 14

(3aR,5R,6S,7R,7aR)-5- (hydroxymethyl)-2-morpholino-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol 290.9

Example 15(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-((cyclopentylamino)methyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol(5)

((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyl4-methylbenzenesulfonate (4)

A solution of(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol(5.0 g, 19 mmol) and triethylamine (3.88 g, 38 mmol) in DMF (50 mL) wastreated with 4-methylbenzene-1-sulfonyl chloride (4.4 g, 23 mmol)overnight at room temperature. The reaction mixture was quenched bywater (100 mL), extracted with dichloromethane (3×80 mL), washed withbrine (3×50 mL), dried over anhydrous magnesium sulfate, andconcentrated under vacuum to give a residue, which was purified by asilica gel column eluted with 1%-5% methanol in dichloromethane to givecrude compound 4 as a light yellow solid (1.0 g). This material wasemployed in the next step without further purification. (ES, m/z) [M+H]⁺415.0.

Step 2

(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-((cyclopentylamino)methyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol(5)

A solution of((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyl4-methylbenzenesulfonate (350 mg, 0.85 mmol) in cyclopentanamine (3 mL)was heated to 50° C. for overnight. The reaction mixture was condensedunder vacuum to give a residue, which was purified by Prep-HPLC with thefollowing conditions [(Agilent 1200 prep HPLC): Column, x-Bridge 19*50mm; mobile phase, Water with 0.05% NH₄OH and CH₃CN (15% CH₃CN up to28.6% in 5 min Flow rate 20 mL/min; Detector, UV 220 nm)] to givecompound 5 as a light yellow solid (30 mg, 11%). (ES, m/z) [M+H]⁺ 328.0;¹H NMR (300 MHz, DMSO+D₂O) 6.25-6.27 (d, J=6.3 Hz, 1H), 3.97 (m, 1H),3.86 (m, 4H), 3.71 (m, 1H), 3.45 (m, 1H), 3.25 (m, 1H), 2.95 (m, 1H),2.75 (m, 1H), 2.55 (m, 1H), 2.23 (m, 2H), 1.56-1.72 (m, 6H), 1.25 (m,2H).

The following example was synthesized according to procedures analogousto the schemes and examples outlined above.

TABLE 3 Example Structure Name MH+ 16

(3aR,5R,6S,7R,7aR)-2-(azetidin- 1-yl)-5- ((cyclopropylamino)methyl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazole-6,7-diol 300.0

Example 17((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate (8)

((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-bis(4-methoxybenzyloxy)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate (7)

To a solution of((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-bis(4-methoxybenzyloxy)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methanol(300 mg, 0.60 mmol) in THF (10 mL) was added NaHMDS (1.1 g, 6.01 mmol),and followed by addition of diethylcarbamic chloride (1.22 g, 9.04 mmol)at room temperature. After stirring for 1 h, the reaction mixture wasquenched with saturated aqueous NH₄Cl (50 mL), extracted withdichloromethane (3×30 mL), dried over MgSO₄, filtered, and concentratedunder reduced pressure to give a crude product 7 (320 mg), which wasused in next step directly without further purification. [M+H]⁺ 600.1

Step 2

((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate (8)

A solution of above crude product (320 mg) in dichloromethane (9 mL) wastreated with CF₃COOH (1 mL) for 2 h at room temperature. The reactionmixture was condensed under vacuum to give a residue, which was purifiedby Prep-HPLC with the following conditions [(Agilent 1200 DeteclPrep-HPLC): Column, SunFire Prep C18; mobile phase, water with 0.03%ammonia and CH₃CN; Detector, UV 220 nm] to afford((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate 8 as a yellow solid (97.5 mg, 51%). (ES, m/z): [M+H]⁺360.0; ¹H NMR (300 MHz, D₂O) δ 6.25 (d, J=6.3 Hz, 1H), 4.25-4.29 (m,1H), 4.07-4.15 (m, 2H), 3.93-3.99 (m, 5H), 3.69-3.74 (m, 1H), 3.54-3.59(m, 1H), 3.16-3.23 (m, 4H), 2.24-2.23 (m, 2H), 1.03 (t, J=7.2 Hz, 6H).

The following examples were synthesized according to proceduresanalogous to the schemes and examples outlined above.

TABLE 4 Example Structure Name MH+ 18

((3aR,5R,6S,7R,7aR)-2-(azetidin- 1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazol-5-yl)methyl dimethylcarbamate 332.019

((3aR,5R,6S,7R,7aR)-6,7- dihydroxy-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazol-5-yl)methyldimethylcarbamate 346.0 20

((3aR,5R,6S,7R,7aR)-6,7- dihydroxy-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH- pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate 374.0 21

((3aR,5R,6S,7R,7aR)-2-(azetidin- 1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2- d]thiazol-5-yl)methyl ethylcarbamate 332.0

Biological Activity

Assay for Determination of K_(I) Values for Inhibition of O-GlcNAcaseActivity

Experimental Procedure for Kinetic Analyses:

Enzymatic reactions were carried out in a reaction containing 50 mMNaH₂PO₄, 100 mM NaCl and 0.1% BSA (pH 7.0) using 2 mM4-Methylumbelliferyl N-acetyl-β-D-glucosaminide dihydrate (Sigma M2133)dissolved in ddH₂O, as a substrate. The amount of purified humanO-GlcNAcase enzyme used in the reaction was 0.7 nM. Test compound ofvarying concentrations was added to the enzyme prior to initiation ofthe reaction. The reaction was performed at room temperature in a96-well plate and was initiated with the addition of substrate. Theproduction of fluorescent product was measured every 60 sec for 45 minwith a Tecan Infinite M200 plate-reader with excitation at 355 nM andemission detected at 460 nM, with 4-Methylumbelliferone (Sigma M1381)used to produce a standard curve. The slope of product production wasdetermined for each concentration of compound tested and plotted, usingstandard curve fitting algorithms for sigmoidal dose response curves.The values for a four parameter logistic curve fit of the data weredetermined.

K_(I) values were determined using the Cheng-Prusoff equation; the Km ofO-GlcNAcase for substrate was 0.2 mM.

Examples 1 to 21 were tested in the above described assay and, with theexception of Examples 2 and 8, exhibited K_(I) values for inhibition ofO-GlcNAcase in the range 0.1 nM-50 μM

Assay for Determination of K_(I) Values for Inhibition ofβ-Hexosaminidase Activity

Experimental Procedure for Kinetic Analyses:

Enzymatic reactions were carried out in a reaction containing 50 mMNaH₂PO₄, 100 mM NaCl and 0.1% BSA (pH 7.0) using 2 mM4-Methylumbelliferyl N-acetyl-β-D-glucosaminide dihydrate (Sigma M2133)dissolved in ddH₂O, as a substrate. The amount of purified human13-hexosaminidase enzyme used in the reaction was 24 nM. Test compoundof varying concentrations was added to the enzyme prior to initiation ofthe reaction. The reaction was performed at room temperature in a96-well plate and was initiated with the addition of substrate. Theproduction of fluorescent product was measured every 60 sec for 45 minwith a Tecan Infinite M200 plate-reader with excitation at 355 nM andemission detected at 460 nM, with 4-Methylumbelliferone (Sigma M1381)used to produce a standard curve. The slope of product production wasdetermined for each concentration of compound tested and plotted, usingstandard curve fitting algorithms for sigmoidal dose response curves.The values for a four parameter logistic curve fit of the data weredetermined.

K_(I) values were determined using the Cheng-Prusoff equation.

When tested in this assay, many of the compounds described hereinexhibited K_(I) values for inhibition of 3-hexosaminidase in the range10 nM to greater than 100 μM.

The selectivity ratio for inhibition of O-GlcNAcase over3-hexosaminidase is defined here as:

-   -   K_(I) (β-hexosaminidase)/K_(I)(O-GlcNAcase)        In general, the compounds described herein exhibited a        selectivity ratio in the range of about 10 to 100000. Thus, many        compounds of the invention exhibit high selectivity for        inhibition of O-GlcNAcase over β-hexosaminidase.

Assay for Determination of Cellular Activity for Compounds that InhibitO-GlcNAcase Activity

Inhibition of O-GlcNAcase, which removes O-GlcNAc from cellularproteins, results in an increase in the level of O-GlcNAcylated proteinin cells. An increase in O-GlcNAcylated protein can be measured by anantibody, such as RL-2, that binds to O-GlcNAcylated protein. The amountof O-GlcNAcylated protein:RL2 antibody interaction can be measured byenzyme linked immunosorbant assay (ELISA) procedures.

A variety of tissue culture cell lines, expressing endogenous levels ofO-GlcNAcase, can be utilized; examples include rat PC-12, and humanU-87, or SK—N—SH cells. Rat PC-12 cells were plated in 96-well plateswith approximately 10,000 cells/well. Compounds to be tested weredissolved in DMSO, either 2 or 10 mM stock solution, and then dilutedwith DMSO and water in a two-step process using a Tecan workstation.Cells were treated with diluted compounds for 24 h (5.4 μL into 200 μL 1well volume) to reach a final concentration of inhibitor desired tomeasure a compound concentration dependent response, typically, ten 3fold dilution steps, starting at 10 μM were used to determine aconcentration response curve. To prepare a cell lysate, the media fromcompound treated cells was removed, the cells were washed once withphosphate buffered saline (PBS) and then lysed for 5 minutes at roomtemperature in 50 μL of Phosphosafe reagent (Novagen Inc, Madison, Wis.)with protease inhibitors and PMSF. The cell lysate was collected andtransferred to a new plate, which was then either coated to assay platesdirectly or frozen −80° C. until used in the ELISA procedure. Ifdesired, the total protein concentration of samples was determined using20 μL of the sample using the BCA method.

The ELISA portion of the assay was performed in a black Maxisorp 96-wellplate that was coated overnight at 4° C. with 100 μL/well of the celllysate (1:10 dilution of the lysate with PBS containing proteaseinhibitors, phosphatase inhibitors, and PMSF). The following day thewells were washed 3 times with 300 μL/well of Wash buffer (Tris-bufferedsaline with 0.1% Tween 20). The wells were blocked with 100 μL/wellBlocking buffer (Tris buffered saline w/0.05% Tween 20 and 2.5% Bovineserum albumin). Each well was then washed two times with 300 uL/well ofwash buffer. The anti O-GlcNAc antibody RL-2 (Abcam, Cambridge, Mass.),diluted 1:1000 in blocking buffer, was added at 100 ul/well. The platewas sealed and incubated at 37° C. for 2 hr with gentle shaking. Thewells were then washed 3-times with 300 uL/well wash buffer. To detectthe amount of RL-2 bound horse-radish peroxidase (HRP) conjugated goatanti-mouse secondary antibody (diluted 1:3000 in blocking buffer) wasadded at 100 μL/well. The plate was incubated for 60 min at 37° C. withgentle shaking. Each well was then washed 3-times with 300 uL/well washbuffer. The detection reagent was added, 100 μL/well of Amplex Ultra REDreagent (prepared by adding 30 μL of 10 mM Amplex Ultra Red stocksolution to 10 mL PBS with 18 μL 3% hydrogen peroxide, H₂O₂). Thedetection reaction was incubated for 15 minutes at room temperature andthen read with excitation at 530 nm and emission at 590 nm.

The amount of O-GlcNAcylated protein, as detected by the ELISA assay,was plotted for each concentration of test compound using standard usingstandard curve fitting algorithms for sigmoidal dose response curves.The values for a four parameter logistic curve fit of the data weredetermined, with the inflection point of the curve being the potencyvalue for the test compound.

Representative data from the binding and cell-based assays describedabove are shown in the following table.

Fluorescence-based Cell-based ELISA hOGA Example # EC₅₀ (nM) Ki (nM) 128 3.3 3 ND 30 5 ND 142 7 165  13 14 ND 289 16 ND 101 18 ND 350

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

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1. A compound of Formula (I) or a pharmaceutically acceptable saltthereof:

wherein each R¹ is independently H or C₁₋₆ acyl; both R² groups arejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ is OR⁴ or NR⁴ ₂; and each R⁴ is independentlyselected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, or carbamoyl.2. The compound of claim 1 wherein R¹ is H or C(O)CH₃;
 3. The compoundof claim 1 wherein NR²² is

each optionally independently substituted from one up to the maximumnumber of substituents with one or more of: fluoro, OH, methyl, or OCH₃.4. The compound of claim 1 wherein R³ is OH, NH(cyclopropyl),NH(cyclopentyl), O(CO)NH(CH₂CH₃), O(CO)N(CH₃)₂, O(CO)N(CH₂CH₃)₂, orO(CO)CH₃.
 5. The compound of claim 1 wherein: R¹ is H or C(O)CH₃; NR² ₂is

; and R³ is OH, NH(cyclopropyl), NH(cyclopentyl), O(CO)NH(CH₂CH₃),O(CO)N(CH₃)₂, O(CO)N(CH₂CH₃)₂, or O(CO)CH₃.
 6. (canceled)
 7. Thecompound of claim 1 wherein the compound is selected from the followinggroup:(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(acetoxymethyl)-2-(azetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diyldiacetate;(3aR,5R,6S,7R,7aR)-2-(3-fluoroazetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-2-(3-hydroxyazetidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(hydroxymethyl)-2-(3-methoxyazetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(hydroxymethyl)-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(acetoxymethyl)-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diyldiacetate;(3aR,5R,6S,7R,7aR)-2-((S)-3-fluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-2-((R)-3-fluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-2-(3,3-difluoropyrrolidin-1-yl)-5-(hydroxymethyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(hydroxymethyl)-2-(piperidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(hydroxymethyl)-2-morpholino-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-((cyclopentylamino)methyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-5-((cyclopropylamino)methyl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate;((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldimethylcarbamate;((3aR,5R,6S,7R,7aR)-6,7-dihydroxy-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldimethylcarbamate;((3aR,5R,6S,7R,7aR)-6,7-dihydroxy-2-(pyrrolidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methyldiethylcarbamate;((3aR,5R,6S,7R,7aR)-2-(azetidin-1-yl)-6,7-dihydroxy-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazol-5-yl)methylethylcarbamate;(3aR,5R,6S,7R,7aR)-5-(hydroxymethyl)-2-(3-methylazetidin-1-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;(3aR,5R,6S,7R,7aR)-5-(hydroxymethyl)-2-(isoxazolidin-2-yl)-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]thiazole-6,7-diol;or a pharmaceutically acceptable salt of any of the foregoing compounds.8. The compound of claim 1 wherein the compound is a prodrug.
 9. Thecompound of claim 1 wherein the compound selectively inhibits anO-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidase (O-GlcNAcase),selectively binds an O-GlcNAcase, selectively inhibits the cleavage of2-acetamido-2-deoxy-β-D-glucopyranoside (O-GlcNAc), or does notsubstantially inhibit a mammalian β-hexosaminidase. 10.-11. (canceled)12. The compound of claim 9 wherein the O-GlcNAcase is a mammalianO-GlcNAcase.
 13. (canceled)
 14. A pharmaceutical composition comprisingthe compound of claim 1 or a pharmaceutically acceptable salt thereof incombination with a pharmaceutically acceptable carrier.
 15. A method ofselectively inhibiting an O-GlcNAcase or of elevating the level ofO-GlcNAc or of treating a condition that is modulated by an O-GlcNAcasein a subject in need thereof, the method comprising administering to thesubject an effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof:

wherein each R¹ is independently H or C₁₋₆ acyl; both R² groups arejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ is OR⁴ or NR⁴ ₂; and each R⁴ is independentlyselected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, or carbamoyl.16.-17. (canceled)
 18. The method of claim 15 wherein the condition isselected from one or more of the group consisting of an inflammatorydisease, an allergy, asthma, allergic rhinitis, hypersensitivity lungdiseases, hypersensitivity pneumonitis, eosinophilic pneumonias,delayed-type hypersensitivity, atherosclerosis, interstitial lungdisease (ILD), idiopathic pulmonary fibrosis, ILD associated withrheumatoid arthritis, systemic lupus erythematosus, ankylosingspondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis ordermatomyositis, systemic anaphylaxis or hypersensitivity response, drugallergy, insect sting allergy, autoimmune disease, rheumatoid arthritis,psoriatic arthritis, multiple sclerosis, Guillain-Barré syndrome,systemic lupus erythematosus, myastenia gravis, glomerulonephritis,autoimmune thyroiditis, graft rejection, allograft rejection,graft-versus-host disease, inflammatory bowel disease, Crohn's disease,ulcerative colitis, spondyloarthropathy, scleroderma, psoriasis, T-cellmediated psoriasis, inflammatory dermatosis, dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria, vasculitis,necrotizing, cutaneous, and hypersensitivity vasculitis, eosinphilicmyotis, eosiniphilic fasciitis, solid organ transplant rejection, hearttransplant rejection, lung transplant rejection, liver transplantrejection, kidney transplant rejection, pancreas transplant rejection,kidney allograft, lung allograft, epilepsy, pain, fibromyalgia, stroke,neuroprotection, Alzheimer's disease, Amyotrophic lateral sclerosis(ALS), Amyotrophic lateral sclerosis with cognitive impairment (ALSci),Argyrophilic grain dementia, Bluit disease, Corticobasal degeneration(CBD), Dementia pugilistica, Diffuse neurofibrillary tangles withcalcification, Down's syndrome, Familial British dementia, FamilialDanish dementia, Frontotemporal dementia with parkinsonism linked tochromosome 17 (FTDP-17), Gerstmann-Straussler-Scheinker disease,Guadeloupean parkinsonism, Hallevorden-Spatz disease (neurodegenerationwith brain iron accumulation type 1), Multiple system atrophy, Myotonicdystrophy, Niemann-Pick disease (type C), Pallido-ponto-nigraldegeneration, Parkinsonism-dementia complex of Guam, Pick's disease(PiD), Post-encephalitic parkinsonism (PEP), Prion diseases (includingCreutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Fatal Familial Insomnia, Kuru, Progressive supercorticalgliosis, Progressive supranuclear palsy (PSP), Richardson's syndrome,Subacute sclerosing panencephalitis, Tangle-only dementia, Huntington'sdisease, Parkinson's disease, Schizophrenia, Mild Cognitive Impairment(MCI), Neuropathy (including peripheral neuropathy, autonomicneuropathy, neuritis, and diabetic neuropathy), Glaucoma, or a cardiacdisorder.
 19. The method of claim 15 wherein the condition selected fromthe group consisting of a neurodegenerative disease, a tauopathy, cancerand stress. 20.-21. (canceled)
 22. The method of claim 18 wherein thecardiac disorder is selected from one or more of the group consisting ofischemia; hemorrhage; hypovolemic shock; myocardial infarction; aninterventional cardiology procedure; cardiac bypass surgery;fibrinolytic therapy; angioplasty; and stent placement.
 23. The methodof claim 15 wherein R¹ is H or C(O)CH₃.
 24. The method of claim 15wherein R³ is OH or OC(O)CH₃.
 25. The method of claim 15 wherein NR₂ is


26. (canceled)
 27. The method of claim 15 wherein said administeringincreases the level of O-GlcNAc in the subject.
 28. The method of claim15 wherein the subject is a human. 29.-30. (canceled)
 31. A method forscreening for a selective inhibitor of an O-GlcNAcase, the methodcomprising: a) contacting a first sample with a test compound; b)contacting a second sample with a compound of Formula (I)

wherein each R¹ is independently H or C₁₋₆ acyl; both R² groups arejoined together with the nitrogen atom to which they are attached toform a ring, said ring optionally independently substituted from one upto the maximum number of substituents with one or more of: fluoro, OH,methyl, or OCH₃; R³ is OR⁴ or NR⁴ ₂; and each R⁴ is independentlyselected from the group consisting of: H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₂₋₆ alkenyl, C₃₋₆ cycloalkenyl, C₂₋₆ alkynyl, C₁₋₆ acyl, or carbamoyl;c) determining the level of inhibition of the O-GlcNAcase in the firstand second samples, wherein the test compound is a selective inhibitorof a O-GlcNAcase if the test compound exhibits the same or greaterinhibition of the O-GlcNAcase when compared to the compound of Formula(I).